Regulation of normal and Leber congenital amaurosis-associated RPE65s

正常和 Leber 先天性黑蒙相关 RPE65 的调节

• 批准号: 8184667
• 负责人: Minghao Jin
• 金额: $ 35.5万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8184667
• 关键词: 11 cis Retinal; 11-cis-Retinol; 26S proteasome; Acyl Coenzyme A; Binding; Biochemical; Biochemical Reaction; Blindness; Cattle; Cells; Childhood; Coenzyme A Ligases; Cyclic GMP; Degenerative Disorder; Development; Enzymes; Esters; Exhibits; Expression Library; Gated Ion Channel; Genes; Goals; Human; Hyperactive behavior; In Vitro; Isomerase; Isomerism; Knock-in Mouse; Knock-out; Knowledge; Lead; Leber' s amaurosis; Light; Link; Membrane; Membrane Proteins; Missense Mutation; Molecular; Mus; Mutant Strains Mice; Mutation; Natural regeneration; Opsin; Pathogenicity; Pathway interactions; Patients; Phenotype; Photoreceptors; Phototransduction; Pigments; Proteins; RPE65 protein; Regulation; Research; Research Project Grants; Retinal Degeneration; Retinal Pigments; Retinoids; Rhodopsin; Role; Screening procedure; Series; Signal Transduction; Site; Structure of retinal pigment epithelium; Testing; Therapeutic Intervention; Ubiquitin; Very Long Chain Fatty Acid; Vision; base; cold temperature; early onset; expression cloning; fatty acid-transport protein; in vivo; inhibitor/antagonist; innovation; loss of function; multicatalytic endopeptidase complex; mutant; novel; photoreceptor degeneration; prevent; response; retinol isomerase; retinyl palmitate; visual cycle

DESCRIPTION (provided by applicant): The visual cycle is a series of enzymatic reactions essential for regenerating 11-cis retinal (11cRAL), which functions as a molecular switch for activating opsin in response to light stimulation. 11cRAL keeps opsin inactive. When light hits the visual pigments, its energy converts the pigments' 11cRAL into all-trans isomer, thus activating opsin. The activated opsin then triggers phototransduction, which converts the light-activated biochemical signal to an electrical energy by closing the cGMP-gated ion channels in the photoreceptors. Since the opsin that lost 11cRAL is no longer responsive to light, 11cRAL must be regenerated via the visual cycle to re-form the light-sensitive visual pigments. The key enzymatic reaction in the visual cycle is the isomerization of the all-trans retinoid to an 11-cis isomer. We have identified RPE65, a retinal pigment epithelium (RPE) specific membrane-associated protein, as the retinoid isomerase. Mutations in the human RPE65 gene have been associated with an early-onset retinal degenerative disease known as Leber congenital amaurosis (LCA). Despite the importance of this protein, the molecular mechanisms that regulate the function of RPE65 are largely unknown. The long-term goals of our research are (1) to define the mechanisms that regulate the enzymatic activity of normal and LCA-associated (LCAA) RPE65s and (2) to develop a novel and effective strategy for rescuing LCAA RPE65s. Identification and characterization of proteins that regulates RPE65 function is the key to defining the mechanisms. By screening of the bovine RPE expression library, we have identified three negative regulators of RPE65. The goal of Specific Aim 1 of this proposal is to elucidate the action mechanisms of the negative regulators in vitro and determine their functional role in the visual cycle in vivo. As a step toward defining the inhibitory mechanisms of RPE65 by the regulators, we will first test if the new regulators bind with RPE65. Next we will test if the new regulator can compete with RPE65 for binding to the substrate of RPE65. We will then determine the role of the new regulator in vision by analyzing the visual cycle-related phenotypes of the mutant mice that lack the new regulator. The goal of Specific Aim 2 of this project is to determine whether the new regulators are involved in the pathological mechanism of the LCAA RPE65 mutations and to define the molecular basis for the pathogenicity of LCAA RPE65s with non-active site missense mutations. The proposed research is innovative because it can establish a new research direction in regulation of isomerase activities of normal and LCAA RPE65s and can lead to the development of a novel rescue strategy for delaying or preventing vision loss and photoreceptor degeneration in patients with LCA that is caused by mutations in the RPE65 gene. PUBLIC HEALTH RELEVANCE: Relevance: Mutations in the human RPE65 gene cause early onset childhood blindness known as Leber's congenital amaurosis (LCA). The proposed research project will increase our understanding of how the function of the normal and LCA-associated mutant RPE65s is regulated. The knowledge gained from this research will facilitate the development of a novel and effective therapeutic intervention to delay or prevent vision loss in patients with LCA.

描述（由申请人提供）：视觉周期是一系列对于再生11张视网膜（11克）必不可少的酶促反应，该反应是一种分子开关，可用于响应光刺激而激活Opsin。 11Cral保持OPSIN不活跃。当光击中视觉颜料时，其能量会将颜料的11克转化为全型异构体，从而激活Opsin。然后，激活的OPSIN触发光转导的光转导，该光转导将光激活的生化信号转换为电能，通过关闭光感受器中的CGMP门控离子通道。由于失去11克的蛋白不再对光的响应，因此必须通过视觉周期再生11克，以重新形成光敏的视觉色素。视觉循环中的关键酶促反应是全反式视网膜类似于11-CIS异构体的异构化。我们已经将RPE65（一种视网膜色素上皮（RPE）特异性膜相关蛋白）确定为视网膜类动物酶。人RPE65基因的突变与一种称为Leber先天性amaurosis（LCA）的早期视网膜退行性疾病有关。尽管该蛋白质很重要，但调节RPE65功能的分子机制在很大程度上尚不清楚。我们研究的长期目标是（1）定义调节正常和LCA相关（LCAA）RPE65S的酶活性的机制，以及（2）制定一种新颖有效的策略来挽救LCAA RPE65S。调节RPE65功能的蛋白质的识别和表征是定义机制的关键。通过筛选牛RPE表达式文库，我们已经确定了RPE65的三个负调节剂。该提案的特定目的1的目的是阐明体外调节剂的作用机制，并确定其在体内视觉周期中的功能作用。作为定义调节器RPE65抑制机制的一步，我们将首先测试新调节剂与RPE65结合。接下来，我们将测试新调节器是否可以与RPE65竞争与RPE65的基板结合。然后，我们将通过分析缺乏新调节剂的突变小鼠的视觉周期相关表型来确定新调节剂在视觉中的作用。该项目的特定目标2的目的是确定新调节剂是否参与LCAA RPE65突变的病理机制，并定义具有非活动位点错过突变的LCAA RPE65的致病性分子基础。拟议的研究具有创新性，因为它可以在调节正常和LCAA RPE65s的异构酶活性方面建立新的研究方向，并可能导致开发一种新型的救援策略，以延迟或预防LCA患者的视力丧失和受感受器退化，这是由RPE65基因中突变引起的。 公共卫生相关性：相关性：人类RPE65基因的突变导致早期发作的儿童失明，称为Leber的先天性症（LCA）。拟议的研究项目将增加我们对正常和LCA相关突变体RPE65S的功能的理解。从这项研究中获得的知识将有助于开发新的有效的治疗干预措施，以延迟或防止LCA患者的视力丧失。